For the biotechnological production of biologically active or therapeutic proteins in mammalian cells, so-called biopharmaceuticals, the corresponding mammalian cells are stably transfected with DNA which codes for the biologically active protein (or its subunits). After the transfection process a pool of millions of differently transfected cells is normally obtained. Therefore the crucial step for the preparation of efficient production cell lines is in the selection and replication of cell clones which on the one hand grow very stably and on the other hand show a high specific productivity of therapeutic protein (product formation etc.). As there are millions of different product-expressing cells, it is critical to be able to analyse a plurality of cells individually with a high throughput and to use automation in order to be able to sort out suitable candidates (single cell clones) which both grow very robustly and also yield high product titres. This process of single cell isolation and subcultivation is known as cloning or recloning.
Transfected cells may be selected by fluorescence-activated cell sorting (FACS) for example, by linking the expression of the therapeutic protein to the expression of a marker protein. For this purpose for example fluorescent proteins and the variants thereof of Aequorea victoria, Renilla reniformis or other species, including but not limited to the red, yellow, violet, green fluorescent proteins or the variants thereof of non-bioluminescent organisms such as e.g. Discosoma sp., Anemonia sp., Clavularia sp., Zoanthus sp. may be co-expressed in a cell together with the therapeutic protein. Conclusions may be drawn from the fluorescence intensity as to the specific productivity and the growth characteristics of the cells.
However, there is the problem of depositing typical recombinant production cells such as mouse myeloma (NS0), hamster ovary (CHO), or hamster kidney cells (BHK), particularly if they are adapted to growth in serum-free suspension cultures, i.e. under modern production-relevant cell culture conditions, individually in culture vessels, e.g. in the wells of microtitre plates, under serum-free culture conditions, and effectively replicating (recloning) them. If only a few cells, for example less than 5 cells, are deposited in a culture vessel under serum-free conditions, these cells cannot replicate at all, or at least cannot replicate efficiently. The reason for this is suspected to be the absence of cell-to-cell contacts, a greater nutrient/growth factor requirement at a lower cell density and/or the absence or very low concentration of diffusible signal and conditioning factors.
In the prior art the problem of serum-free single cell cloning in the above-mentioned recombinant production cells is avoided by generating cell clones by the limited dilution method. In this, a minimum of 5 to 10 cells are seeded in serum-free medium in a culture dish and then subpassaged by repeated dilution cloning in order to obtain, in statistical terms, a culture consisting of genetically identical cells (method=limited dilution). On the one hand this method of recloning is time-consuming and on the other hand it usually leads to genetically heterogeneous mixed cultures, as the process is based on a statistical calculation and not on actual cultivation of individually deposited genetically identical cells. These heterogeneous mixed cultures are generally characterised by limited robustness with respect to fermentation and a heterologous expression profile.
Alternatively, at present, single cell clones of production-relevant cell lines can only be generated by the individual depositing of serum-adapted adherent cells. Thus, Meng et al., (Meng Y G., et al., Gene 2000, 242, 201-207) mention, for example, a method of depositing individual, adherently growing CHO cells in serum-containing medium. The method described by Meng et al., however, has major disadvantages; i.e., because of the adherence of the cells, the laborious enzymatic detachment of the cells from the substrate (trypsin treatment) may lead to considerable cell damage and changes in the growth characteristics and in the productivity of the recloned cells. Moreover, the correspondingly obtained individual clones then have to be adapted to serum-free growth in suspension culture, which is normally a time-consuming operation and affects the productivity of the cells and the product quality (cf. on this subject, inter alia, Kaufmann H. et al., Biotechnology and Bioengineering 2001, 72, 592-602; Mueller et al., Biotechnology and Bioengineering 1999, 65, 529-532).
By using nutrient cells, also known as feeder cells, in the cultivation of adherently growing cells, it is possible to influence the growth characteristics of cells for the better or, for some types of cell, to replicate them under cell culture conditions for the first time. Examples include human-mouse or mouse-mouse hybridoma cells (Hlinak A. et al., Folia Biologica (Praha) 1988, 34, 105.117, U.S. Pat. No. 5,008,198), primary keratinocytes (Rheinwald, J G and Green, Cell 1975, 6, 331-344; WO 9954435), stem cells (Williams R L., et al., Nature 1988, 336, 684-687) and various tumour cells (Wee Eng Lim et al., Proteomics 2002, 2, 1187-1203; Rexroad et al., Molecular Reproduction and Development, 1997, 48, 238-245; Peng et al., Biotechnology and Bioengineering, 1996, 50, 479-492; Grigoriev et al., Analytical Biochemistry, 1996, 236, 250-254; Sanchez et al., Journal of Immunological Methods, 1991, 145, 193-197; Butcher et al., Journal of Immunological Methods, 1988, 107, 245-251; Long et al., Journal of Immunological Methods, 1986, 86, 89-93; Shneyour et al., Plant Science Letters, 1984, 33, 293-302; Pintus et al., Journal of Immunological Methods, 1983, 61, 195-200; Brodin et al., J Immunological Methods, 1983, 60(1-2), 1-7). Feeder cells are usually cells the growth of which has been chemically or physically arrested, which have lost their capacity for cell division as the result of a special pre-treatment but otherwise still remain vital for about 2 to 3 weeks on average. Feeder cells are thus still capable of releasing growth-promoting factors into the medium and can thus promote the initial growth of non-arrested cells or even make this growth possible, in the case of various primary cells. For this purpose the feeder cells are plated out in a culture dish as a so-called monolayer. Then the adherently growing cells which are to be cultivated are plated out on or between the feeder cells and cultivated under standard conditions. Feeder cells may be prepared for example by irradiation or treatment with mitomycin C (azirino[2′,3′:3,4]pyrrolo[1,2-a]indole-4,7-dione,6-amino-8-[[(aminocarbonyl)oxy]methyl]-1,1a,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl-, [1aR-(1a.alpha., 8.beta., 8a.alpha., 8b.alpha.)]-(9Cl) (Butcher et al., Journal of Immunological Methods, 1988, 107, 245-251)). Primary cells such as spleen cells, fibroblasts, blood cells (Morgan, Darling; Kultur tierischer Zellen [culture of animal cells]. Spektrum Akademischer Verlag 1994, p. 115f) and macrophages (Hlinak A. et al., Folia Biologica (Praha) 1988, 34, 105.117) have been described in feeder cell systems. In connection with the production of antibodies in hybridoma cells the use of feeder cells and FACS-based cell selection was described. Hlinak et al. (1988) for example describe a recloning efficiency of 33 to 57% in the recloning of adherently growing hybridoma cells starting from two (2) cells per culture dish. The hybridoma cells used were adherently growing cells adapted to serum-containing medium.
When using heterologous, particularly human feeder cells for generating production cells there is a considerable risk of contamination by pathogens such as, for example, viruses, bacteria or mycoplasms. Moreover, many of the (primary) feeder cells described require serum-containing medium, which firstly increases the risk of contamination and secondly has the drawback that production cells which have laboriously been adapted to serum-free growth have to be re-adapted.
When using heterologous feeder cells, it is generally necessary to counter-select the feeder cells. Production cells are generally subject to a selection pressure, e.g. as a result of the use of additives to the medium (antibiotics such as G418) and/or incomplete media (absence of hypoxanthine, thymidine). This selection pressure makes it possible to select cells, for example, which have absorbed and integrated the corresponding genetic information for a recombinant protein. The medium thus produced which is adapted to the production cell critically influences the growth of the feeder cells and together with the otherwise incompatible production medium leads to a very rapid dying off of the feeder cells. As a result, the function of the feeder cells is no longer guaranteed.